Food allergy is a major public health problem in the Western world that is increasing in prevalence at a very significant rate, particularly among children. Moreover, our understanding of the basic immunological mechanisms that lead to these conditions remains poor, in part because our methodologies to study these problems are relatively crude. The overall goal of this research is to develop an assay for characterizing allergen-specific T cells using a novel microtechnology that allows high-throughput, sensitive profiling of individual lymphocytes. We will apply this new assay to evaluate the frequency of food-specific T cells in food- allergic individuals, and then to monitor the evolution of the functional diversity of food-specific T cells in children undergoing oral milk desensitization. Our technology addresses two challenges that significantly impede detailed immunological studies of allergen-specific T cells in children: the sensitivity required to detect functional responses from individual clones and the ability to handle the small numbers of cells that are available in clinical samples from children. This project is a collaboration between the Love lab (MIT) with expertise for simultaneous parallel analyses of secretory products from >10^5 single cells and the Umetsu lab (Children's Hospital) for clinical analysis and immunological assessments of allergy patients. This interdisciplinary team is unique and innovative in that it combines substantial expertise in both process engineering and human immunology to address the challenges of understanding and treating food allergies. Together, this translational approach will improve our knowledge of the immunological basis of food allergy and tolerance, and of the role of immune deviation, tolerance and deletion of allergen-specific T cells. The specific aims of this project are: 1) to develop an assay to evaluate food-specific T cells using a single-cell microtechnology and 2) to investigate the specific immunological mechanisms by which oral tolerance develops. Establishing quantitative analytical tools may also lead to new clinical diagnostics that complement challenge testing or skin-prick testing, and that enable routine immunological monitoring of the efficacy of other interventional studies. Food allergies are an increasing burden on public health, especially among our children, but the mechanisms by which existing treatments for desensitization operate are not well understood. This project will develop and apply a novel technology based on microfabricated devices to examine the biological mechanisms that govern rapid desensitization to milk by oral treatment. The results will be important for evaluating the utility of this therapy, and the development of the underlying technology used in this research may also lead to new diagnostics for monitoring allergy treatments.